We recently discovered that eukaryotic cells contain enzymes that specifically hydrolyze phosphodiester bonds that link the side chain of tyrosine to the 3'-end of DNA. To test our hypothesis that these enzymes function to repair topoisomerase I dead-end covalent complexes, we are carrying out a genetic and biochemical characterization of these enzymes. Using a partially purified enzyme from budding yeast, S. cerevisiae, the specificity for the tyrosine-DNA bond has been confirmed by showing inactivity on 3'-terminal phosphomonoesters and 3'-terminal phosphoglycolic acid diesters. A strain of yeast that shows low tyrosine-DNA phosphodiesterase activity has been isolated. Biochemical analysis of extracts shows that the defect is most prominent on single-strand substrates and a genetic analysis indicates that the enzyme defect is caused by a single mutation. The effect of this mutation on topoisomerase metabolism is under study. Collaborative projects have been initiated in which the abundance of enzymes putatively involved in topoisomerase repair is evaluated in wild-type cell lines and mutant lines that have altered sensitivity to topoisomerase poisons. Initial results suggests that the latter contain increased levels of 3'-phosphatase, an activity thought to act downstream of the tyrosine-DNA phosphodiesterase.